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P Ascher

Publications and source records attributed to P Ascher.

At least 37 records · Page 2Linked to original sources
PubMed1990

Voltage-dependent block by intracellular Mg2+ of N-methyl-D-aspartate-activated channels.

The N-methyl-D-aspartate (NMDA)-activated channel, which is known to be blocked by extracellular Mg ions, is shown also to be blocked by intracellular Mg ions. The block by intracellular Mg can be explained by assuming that Mg ions from the intracellular side enter the membrane electrical field before binding to the blocking site. The dissociation constant of the binding site for intracellular Mg is 8 mM at 0 mV, which is close to the value previously calculated for the extracellular Mg blocking site. The unbinding rates of intracellular and extracellular Mg are different, and their effects are additive, suggesting that the corresponding binding sites are distinct. Both blocks occur at physiological concentrations of Mg, making the NMDA-activated channel a bidirectional rectifier.

Animals↗
PubMed1990

Competitive antagonists and partial agonists at the glycine modulatory site of the mouse N-methyl-D-aspartate receptor.

1. Kynurenate (Kyn), 7-chlorokynurenate (7-Cl-Kyn), 3-amino-1-hydroxypyrrolid-2-one (HA-966) and D-cycloserine are known to bind to the glycine site that modulates the N-methyl-D-aspartate (NMDA) response of vertebrate central neurones. The effects of these compounds were investigated with patch-clamp and fast-perfusion techniques on mouse cortical neurones in primary culture in an effort to establish whether they act as antagonists, partial agonists and/or inverse agonists of glycine. A fast drug application method allowed the study of both steady-state and transient responses. 2. The analysis of steady-state responses indicates that the main effects of Kyn and 7-Cl-Kyn are those expected from competitive antagonists of glycine, with a dissociation constant of 15 microM for Kyn, and of 0.3 microM for 7-Cl-Kyn. Concentration jumps indicate that at all concentrations of glycine, and in particular in the absence of added glycine, the blockade by Kyn and 7-Cl-Kyn develops at a rate which is close to the rate of dissociation of glycine from its binding site and is independent of antagonist concentration. 3. The main effects of D-cycloserine and of HA-966 are those of partial agonists of high and low efficacy, respectively. In the absence of added glycine, D-cycloserine always produced a potentiation, while HA-966 produced either a potentiation or an inhibition. This can be explained by assuming the presence of a variable level of contaminating glycine. With both D-cycloserine and HA-966, concentration jumps produced biphasic relaxations in which the onset rate of the slow component was, here again, close to the rate of dissociation of glycine from its binding site. 4. These results can be interpreted by assuming that (1) Kyn and 7-Cl-Kyn are competitive antagonists of glycine, (2) HA-966 and D-cycloserine are partial agonists, (3) in the absence of added glycine some glycine is present in the extracellular solution and (4) the response in the total absence of glycine is very small or negligible.

Action Potentials↗
PubMed1988

Patch-clamp recording of amino acid-activated responses in "organotypic" slice cultures.

Patch-clamp recording techniques were used to study the properties of amino acid-activated channels in cultured "organotypic" slices from rat cerebellum and hippocampus. Hippocampal pyramidal cells responded to the three main glutamatergic agonists, N-methyl-D-aspartate (N-Me-D-Asp), quisqualate, and kainate, whereas Purkinje cells responded only to quisqualate and kainate. Analysis of single-channel events recorded in outside-out patches from hippocampal neurons showed large conductance events (50 pS), which occurred more frequently in the presence of glycine. These events could be produced by N-Me-D-Asp and also, at low frequency, by quisqualate. On the other hand, 50-pS events were never observed in Purkinje neurons. This supports the hypothesis that N-Me-D-Asp and "non-N-Me-D-Asp" receptors are distinct molecular entities. Comparison of whole-cell and outside-out patch recordings from Purkinje cells revealed a clear spatial segregation of gamma-aminobutyric acid (GABA) and glutamate receptors: although GABA receptors are found at high density in somatic membrane, quisqualate and kainate receptors are mostly extrasomatic. The results show that organotypic slice cultures are amenable to patch-clamp methods. They also show that, in these cultures, amino acids receptors have specific distribution patterns according to cell type and to region within a cell.

Action Potentials↗
PubMed1988

N-methyl-D-aspartate-activated channels of mouse central neurones in magnesium-free solutions.

1. The whole-cell and outside-out configurations of the patch-clamp method were used to investigate the properties of the channels activated by N-methyl-D-aspartate (NMDA channels) in mouse central neurones in culture. Recording was made in Mg2+-free solutions. 2. In the whole-cell recording mode the currents induced by both NMDA and L-glutamate were accompanied by a large increase in noise. In both cases the noise power spectra were well fitted by single Lorentzian functions and the corresponding mean time constant, tau, was about 6 ms at room temperature. The single-channel conductance, gamma n, estimated from the ratio of the noise variance to the total current, varied between 22 and 40 pS. 3. Endogenous amino acids known to activate NMDA receptors (L-glutamate, L-aspartate, L-cysteine sulphinate and quinolinate) as well as exogenous NMDA agonists such as ibotenate and trans-2,3-piperidine dicarboxylate (trans-PDA) all produced similar responses. In particular, analysis of the current noise yielded tau values between 4 and 8 ms in all cases. 4. NMDA responses were antagonized by 2-amino-5-phosphonovalerate (APV) without any effect on gamma n or tau values measured by noise analysis; NMDA responses were also diminished by D-alpha-aminoadipate and cis-2,3-piperidine dicarboxylate. 5. In outside-out patches, it was observed that the single-channel current amplitude varies linearly as a function of membrane potential between -80 and +60 mV. The reversal potential is near 0 mV. NMDA channels are permeable to Na+, K+ and Cs+, but blocked by choline. The single-channel conductance, gamma e, varies between 40 and 50 pS at room temperature. 6. The NMDA channels open in bursts of short openings interrupted by brief closures. At -60 mV, the closures had a mean duration, tc, of 0.4 +/- 0.2 ms. The mean channel open time, to, was 5.9 +/- 1.0 ms for NMDA and 5.3 +/- 1.7 ms for L-glutamate. The mean burst duration, tb, was 10.5 +/- 0.7 ms for NMDA and 8.5 +/- 2.0 ms for L-glutamate. 7. When the temperature was increased between 14 and 24 degrees C, the NMDA channel conductance increased with a Q10 of 1.6 while the mean open time decreased with a Q10 close to 2. 8. The NMDA channel showed, in addition to the 'main' conductance state (40-50 pS), smaller conductance states of 15 and 35 pS.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1988

Quisqualate- and kainate-activated channels in mouse central neurones in culture.

1. Quisqualate- and kainate-induced currents were recorded in mouse central neurones in culture, using both 'whole-cell' and 'outside-out' configurations. Experiments were made at room temperature. 2. Both quisqualate- and kainate-induced currents invert at 0 mV when the extracellular and intracellular solutions contain similar concentrations of monovalent cations. The changes of reversal potential produced by changes in the monovalent cation concentrations are consistent with the hypothesis that both agonists activate channels selectively permeable to cations. 3. The spectral analysis of the quisqualate- and kainate-induced currents recorded in the whole-cell mode indicates that the main component of the quisqualate noise has a time constant of 10-15 ms while the main component of the kainate noise has a time constant of 2-3 ms. 4. In outside-out patches most of the quisqualate-induced current was carried by channels with a conductance of about 8 pS. A small fraction of the quisqualate-induced current appears to be carried by two other channels: the 'NMDA channel' (40-50 pS) (Ascher, Bregestovski & Nowak, 1988) and a 'fast' channel, with a conductance of 15-35 pS, which was not activated by low concentrations of L-glutamate or by NMDA (N-methyl-D-aspartate). 5. Most of the kainate-induced current can be attributed to a channel characterized by a conductance of about 4 pS. Here again, outside-out patches revealed the presence of an additional channel, with a conductance of about 20 pS. 6. The results are consistent with the notion that there are at least three distinct receptors for excitatory amino acids, each preferentially activated by either NMDA, quisqualate or kainate, each opening channels with multiple conductance states. Other possibilities are discussed.

Animals↗
PubMed1988

The role of divalent cations in the N-methyl-D-aspartate responses of mouse central neurones in culture.

1. Single-channel currents activated by N-methyl-D-aspartate (NMDA) agonists were analysed in the presence of various extracellular concentrations of divalent cations in outside-out patches from mouse neurones in primary culture. 2. In nominally Mg2+-free solutions the opening and closing of the channels leads to rectangular current pulses, the mean duration of which varies little with membrane potential. After addition of Mg2+, the single-channel currents recorded at negative potentials appear in bursts of short openings separated by brief closures. 3. The duration of the short openings decreases with increasing Mg2+ concentration, while the duration of the short closures is independent of the Mg2+ concentration. Depolarization increases the duration of the short openings and decreases the duration of the short closures. 4. The dependence of the burst structure on the Mg2+ concentration and on membrane potential is compatible to a first approximation with a model in which Mg2+ ions enter the open channel and block it by binding at a deep site. A better approximation requires, however, additional assumptions such as Mg2+ permeation and/or interactions between Ca2+ and Mg2+. 5. Increasing the extracellular Ca2+ concentration from 1 to 100 mM produces three effects on the currents flowing through NMDA channels. It shifts the reversal potential towards a positive value (+30 mV); it reduces the outward current flowing through the NMDA channels at very positive potentials; it reduces the inward current flowing at negative potentials. 6. The interpretation of the effects of Ca2+ appears to require three hypotheses: that Ca2+ permeates the NMDA channel, that there exists a significant surface potential at the entrance of the NMDA channel in physiological solutions and that both Ca2+ and monovalent cations bind to the channel, binding being stronger in the case of Ca2+ ions. 7. While Co2+ and, to a lesser extent, Mn2+ mimic the effects of Mg2+ on the NMDA channel, Ca2+, Ba2+ and Cd2+ do not. The distinction between Mg2+-like and Ca2+-like divalent cations corresponds to a difference in the speed of exchange of the water molecules surrounding the cations in solutions. Thus, it is possible that permeation occurs for all the divalent cations, but is slower for those which are slowly dehydrated.

Animals↗
PubMed1987

Ionic channels in mouse astrocytes in culture.

We observed Na, K, and Cl voltage-dependent currents in a patch-clamp study of mouse brain astrocytes. In whole-cell recordings, depolarizations activated inward currents that were identified as Na currents since they were blocked by TTX, although complete block required high concentrations (greater than 1 microM). The corresponding single-channel Na currents were observed in outside-out patches. The channels were opened by a depolarizing pulse applied from a holding potential identical to the resting potential (-70 to -80 mV). Therefore, they may be considered functional Na channels. After addition of veratridine and an alpha-scorpion toxin, the decay of Na currents in whole-cell recordings was slower than observed under control conditions. At the single-channel level, the channels appeared to open in bursts. Depolarization did not increase the duration of the bursts, but inside each burst, increased the time spent in the open state. The K currents observed in the whole-cell recording mode were separated into inactivating and noninactivating currents. The inactivating current resembled the A current in its kinetics, its insensitivity to tetraethylammonium, and its sensitivity to 4-aminopyridine. At the single-channel level, at least 3 classes of K channels were observed at steady depolarized potentials. They resembled the K channels found in chromaffin cells by Marty and Neher (1985). Large conductance channels (385 pS) activated around 0 mV were identified as Cl channels.

4-Aminopyridine↗
PubMed1983

Effects of various cations on the slow K+ conductance increases induced by carbachol, histamine and dopamine in Aplysia neurones.

A study was made of the effects of various cations other than K+ on three K+ conductance increases induced by carbachol, histamine and dopamine in an identified group of Aplysia neurones: the 'A' neurones of the cerebral ganglion. The 3 responses were sensitive to alterations of both the extracellular and the intracellular concentrations of Na+ and Ca2+. In particular, they could be reduced markedly by: (a) lowering [Na]0 (replacing NaCl by either Tris-HCl, glucosamine chloride, MgCl2 or sucrose); (b) increasing [Na]i (by intracellular injection of Na+, or by blockade of the Na+-K+ pump); (c) increasing the extracellular divalent cation concentration; or (d) increasing [Ca]i4. Some of the effects of Na+ and divalent cations appear to occur on reaction steps common to the three K+ responses, while others probably imply reaction steps specific to one of the systems, since they differ according to the agonist used. The sensitivity to Na+ and Ca2+ of slow inhibitory responses due entirely to an increase in K+ conductance must be taken into account in the interpretation of some slow hyperpolarizing responses previously assumed to involve changes in Na+ conductance.

Animals↗
PubMed1982

Interactions between three slow potassium responses controlled by three distinct receptors in Aplysia neurones.

1. A voltage clamp study was made of the K(+) permeability increases produced in certain identifiable neurones of the cerebral ganglion of Aplysia by any one of three distinct agonists (carbachol, histamine and dopamine).2. The three K(+) responses involve three distinct receptors, as shown by the selective effects of reversible antagonists (Gruol & Weinreich, 1979a) as well as by the differential, irreversible effects of trimethyloxonium (TMO) ions.3. Prolonged exposure of the neurones to one of the agonists reduces the response to the same agonist (desensitization) and, over the low concentration range, doubling the concentration of an agonist leads to supra-linear summation (potentiation).4. Prolonged exposure of the neurones to one of the agonists also reduces the response to the other agonists (cross-desensitization) and combined application of two agonists reveals cross-potentiation.5. The time course of desensitization (onset and decay) was the same for the histamine and carbachol responses and, except at very high concentration, was indistinguishable from that of cross-desensitization. Likewise, potentiation was similar in the two agonist systems and did not differ significantly from cross-potentiation.6. The results can be interpreted by assuming that the responses to the three agonists involve specific steps followed by common reaction steps, and that some of the common reaction steps control both potentiation and desensitization.7. The responses to carbachol and histamine differ in their voltage sensitivity. This suggests that one or more of the specific steps are voltage-sensitive.8. Although an increase of the intracellular Ca(2+) concentration can itself open K(+) channels, and also inhibit the responses to the three agonists, an increase of internal Ca(2+) does not appear to play an important role either in the development of the response or in the desensitization process.

Acetylcholine↗
PubMed1979

Studies on the mechanism of action of acetylcholine antagonists on rat parasympathetic ganglion cells.

The mode of action of ACh antagonists on the parasympathetic neurones of the submandibular ganglion of the rat was studied by means of a two-micro-electrode voltage-clamp technique. The currents produced by various agonists (carbachol, ACh, suberylcholine) were studied in steady state and after voltage steps, before and after perfusion of various antagonists. 2. For three antagonists (tubocurarine, hexamethonium, decamethonium) the blocking action increases with hyperpolarization. For three other antagonists (surugatoxin, trimetaphan, mecamylamine) the effects observed at low concentrations appear to be independent of membrane potential, although in some cases voltage dependence of the block was observed for mecamylamine. 3. The blocks the 'open' channel-reception complex. The block produced by tubocurarine, hexamethonium and decamethonium increases with the agonist concentration, an observation which supports a 'sequential' scheme in which the antagonist blocks the 'open' channel-receptor complex. The block produced by trimetaphan and mecamylamine decreases slightly with increased agonist concentration, which in turn suggests that these two compounds are competitive antagonists, preventing binding of the agonists to the closed channel-receptor complex. 4. In the cases where the block is voltage dependent, voltage jumps trigger slow relaxations which are not present in control conditions. In the case of tubocurarine and hexamethonium, the relaxation following a hyperpolarizing voltage jump corresponds to a decrease in conductance. In the case of decamethonium, the slow relaxation is in the opposite direction. 5. The slow relaxations observed with tubocurarine and hexamethonium are speeded by an increase of the antagonist concentration; the slow relaxations observed with decamethonium are slowed by an increase of the decamethonium concentration. 6. The steady-state observations and the relaxations can be interpreted in terms of a scheme in which tubocurarine, hexamethonium and decamethonium act mainly by blocking the channels opened by the cholinergic agonists. 7. The two types of slow relaxation are those predicted if tubocurarine and hexamethonium dissociate slowly from the channel, and decamethonium rapidly. 8. An additional effect of tubocurarine is described, which consists of a potentiation of the rising phase of the response to an ionophoretic pulse. Possible mechanisms of this effect are discussed.

Acetylcholine↗
PubMed1978

Life time and elementary conductance of the channels mediating the excitatory effects of acetylcholine in Aplysia neurones.

1. The excitatory effects of acetylcholine (ACh) on an identified group of Aplysia neurones have been studied under voltage clamp in an attempt to measure the average life time. tau, of the channels opened by ACh and the elementary current, iel, flowing through these channels. The value of tau was determined both from spectral noise analysis and from current relaxations after voltage steps. Both methods lead to similar values. iel was calculated from the ratio of the variance of the ACh induced noise to the mean ACh induced current. 2. tau is increased by hyperpolarization, or by lowering the temperature. At 12 degrees C, tau = 27 msec AT -80 MV, tau = 17 msec at mV. tau is about 5 times smaller at 21 degrees C than at 12 degrees C. 3. iel increases linearly with hyperpolarization. At -80 mV, in Tris-buffered sea water, the mean value of iel was 0.8 X 10)-12) A at 12 degrees C. At 21 degrees C, this value was multiplied by 1.8. 4. The estimate of the ACh reversal potential Erev obtained by extrapolation of the relation between iel and the membrane potential V was + 30 mV. The estimate obtained from the analysis of the instantaneous current changes produced by voltage steps was + 15 mV. The difference between the two values appears to be due to the development of a K curent activated by the entry of Ca into the cell during the ACh response. This current introduces an error in opposite directions into the two estimates of Erev, which can therefore be assumed to be intermediate between + 15 and + 30 mV. An assumed value of + 20 mV yields an elementary conductance of 8 X 10(-12) omega-1 at 12 degrees C in Tris-buffered sea water. 5. The total ACh induced current measured in steady-state conditions increases more with hyperpolarization than does iel. The difference can be entirely accounted for by the fact that hyperpolarization increases tau. 6. When carbachol or tetramethylammonium is applied instead of ACh, the value of iel is identical to that found with ACh, but tau is slightly shorter (about 75%). 7. Inward ACh induced currents can still be observed in solutions where all Na has been replaced by Cs, Mg, or Ca. 8. iel increases when Na is replaced by Cs; it decreases when Na is replaced by Mg or Ca. In all Na-free solutions, tau is larger than in Na sea water: the lengthening of tau is largest for Ca sea water, smallest for Cs sea water. An interpretation of these changes of gamma is proposed. This interpretation may also account for the voltage sensitivity of gamma in normal sea water. 9. Partial replacement of NaCl by TrisCl strikingly reduces the ACh induced current. gamma is not modified by Tris substitution, and the reduction of the total current is entirely accounted for by a steep decrease of iel. Tris does not seem to affect the pore opening and closing processes, but to block the ACh controlled channel.

Acetylcholine↗
PubMed1978

The mode of action of antagonists of the excitatory response to acetylcholine in Aplysia neurones.

1. The mode of action of various antagonists of acetylcholine (ACh) excitatory effects on Aplysia neurones was studied under voltage clamp. ACh was applied by iontophoresis whereas antagonists were applied in the bath. Tubocurarine and hexamethonium were the most thoroughly studied compounds. 2. The 'elementary current', calculated as the ratio of the variance of the ACh noise to the mean ACh induced current, was not modified by any of the antagonists tested. 3. The evolution of the ACh induced current after a voltage jump, which is normally described by a single exponential, was modified by all the antagonists tested. A common feature of the modified relaxations was the appearance, over a certain concentration range of the antagonist, of two successive and opposite exponential components. 4. The characteristics of the composite relaxations depend on the antagonist. For a given antagonist they vary with membrane potential, ACh concentration, and antagonist concentration. 5. The noise power spectra of the ACh induced current showed changes consistent with those of the relaxations. 6. In the absence of antagonists, the current induced by a steady application of ACh increases linearly with hyperpolarization. In the presence of antagonists, the I-V curve shows a marked curvature, indicating a proportionally larger reduction of the ACh response at more negative membrane potentials. 7. The voltage sensitivity of the blocking action of hexamethonium and decamethonium is noticeably stronger than that of monovalent antagonists. 8. A model is proposed which accounts for the observed effects. It assumes that the antagonists studied bind perferentially to the 'activated' ACh-receptor complex, and convert it to a non-conducting state. Kinetic constants can be calculated for this reaction; e.g. for curare, at 12 degrees C and -80 mV, the dissociation and association constants were estimated at 0.1 sec-1 and 4 X 10(5) M-1 sec-1. 9. Partial replacement of the extracellular Na by Tris modifies the relaxations observed in the presence of hexamethonium. Hexamethonium appears less effective in the presence of Tris, which supports the hypothesis that the binding site of the antagonists is linked to the ionic channel.

Acetylcholine↗